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临床上,放疗是治疗肿瘤的主要且有效的手段,但是射线在杀伤肿瘤细胞的同时会对患者机体带来骨损伤[1-2]。临床数据表明肿瘤放疗可导致骨组织减少8%~23%[3]。长期的放疗可以导致患者产生骨密度减少、骨质疏松、骨变薄、骨折、骨坏死等骨损伤[4]。小鼠全身受到2 Gy照射后,骨密度明显减少,进一步的研究结果表明小鼠全身受2 Gy照射后,早期会出现破骨细胞数量的增加和功能的提升[5-6]。
MC3T3-E1细胞系是源于C57BL/6小鼠颅盖骨的成骨细胞系,是研究成骨细胞的良好模型[7]。Notch信号转导通路对成骨细胞的增殖、分化和功能有重要影响[8-9]。转基因小鼠实验结果表明,Notch通路对成骨细胞的分化具有抑制作用[10]。Notch信号通路胞内结构域(Notch intracellular domain,NICD)是Notch信号通路中重要的信号分子,其在Notch信号通路中起关键的承上启下作用。
我们利用RNA干扰技术抑制NICD表达建立稳定抑制NICD表达的MC3T3-E1细胞株,并于体外诱导分化为前体成骨细胞和成骨细胞。在辐射损伤后,对前体成骨细胞和成骨细胞的生长和功能基因的变化进行了观察。
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MC3T3-E1细胞系购买于中国医学科学院基础医学研究所。DMEM培养基、胎牛血清(美国Gibco公司);Opti-MEM培养基、Lipofectamine 2000转染试剂、Trizol试剂、M-MLV逆转录酶试剂盒、实时定量PCR(real-time quantitative PCR,qRT-PCR)试剂盒(美国Invitrogen公司);G418、Roche Cell Proliferation ELISA、5-溴-2’-脱氧尿嘧啶核苷(5-bromo-2’-deoxyuridine,BrdU)(colorimetric)试剂盒(德国Sigma-Aldrich公司);蛋白浓度测定试剂盒(上海生工生物工程有限公司);鼠源β-actin单抗(日本Santa Cruz Biotechnology公司);兔源NICD多抗(美国Millipore公司)。
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137Cs γ射线放射源(加拿大Gamma Ⅱ40,吸收剂量率0.99 Gy/min);CFX96TouchTM Real time PCR仪、ChemiDoc TMXRS+成像系统、Mini-PROTEAN®Tetra电泳槽、Sub-Cell®GT Cell水平小型电泳槽、Trans-Blot®半干转印槽、Gene Pulser MXcellTM电穿孔系统(美国Bio-Rad公司);M200酶标仪(瑞士Tecan公司);Nanodrop 2000 UV-Vis分光光度计、HEPA Class100二氧化碳培养箱(美国Thermo公司);倒置显微镜(上海蔡康光学仪器厂);-80℃超低温冰箱(美国Thermo Forma公司);4℃冰箱(广东容声电器股份有限公司);-20℃冰箱(日本三洋电器);高速台式离心机(上海嘉鹏科技有限公司);5430R小型台式高速冷冻离心机(德国Eppendorf);低速离心机(安徽中科中佳科学仪器有限公司);微量恒温器(绍兴市卫星医疗设备制造有限公司)。
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将MC3T3-E1细胞系细胞加入含10%胎牛血清、青霉素(100 U/mL)和链霉素(100 pg/mL)的DMEM完全培养液中,置于37℃、5% CO2、饱和湿度恒温培养箱中培养,每2 ~ 3 d更换培养液1次。待细胞贴壁生长至70% ~ 80%融合时,弃去培养液,PBS清洗,消化,室温离心5 min(1000 r/min,离心半径5 cm),用于细胞实验。
MC3T3-E1细胞贴壁生长至70%~80%融合时,根据文献[11]和[12]将培养液更换为成骨细胞分化培养液(osteoblast differentiation medium,OBDM)培养,同样每2 ~ 3 d更换培养液1次,培养8 d左右则可分化为前体成骨细胞,培养18 d左右分化为成熟的成骨细胞。OBDM完全培养液中含有10-8 mol/L β甘油磷酸钠、50 μg/mL维生素C和10-8 mol/L地塞米松。
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将培养5 d的前体成骨细胞和培养15 d的成骨细胞以137Cs γ射线放射源分别进行2 Gy照射。同时,参照文献[13],继续培养3 d后,将细胞样品用于实验分析。
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用Trizol法提取细胞样品中的RNA。根据M-MLV逆转录试剂盒说明书进行逆转录。逆转录后的cDNA分装,置于-20℃冰箱中保存备用。
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选取NICD序列1691作为干扰位点,干扰序列设计、合成和表达载体pGPH1/GFP/Neo构建由上海吉码公司完成。NICD的RNA干扰序列正反向分别是5’-CACCGCATATGTATGCCAGGTTATGT-TCAAG-3’和5’-AGACATAACCTGGCATACATATG-CTTTTTTG -3’。
将生长良好的MC3T3-E1细胞置于96孔板中,每孔100 μL,含细胞1×104个,培养过夜。每孔换用无抗生素的完全培养基100 μL,培养过夜。将4 μg的NICD的RNA干扰序列载体DNA加入250 μL无血清的Opti-MEM培养基中。加入5 μL Lipofectamine 2000于250 μL相同的无血清Opti-MEM培养基中。将上述两种液体混匀,室温下放置30 min,使cDNA和Lipofectamine 2000形成复合物。弃去转染培养液,将之前形成的复合物加到96孔板中,每孔100 μL,培养24 h后,用G418进行筛选,成功转染后的细胞将具有G418抗性。倒置荧光显微镜下观察结果。
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根据colorimetric试剂盒说明书进行操作,将照射后3 d的MC3T3-E1细胞离心(1000转/min,离心半径为5 cm)弃去培养基,用PBS洗3遍,每孔加入100 μL已经加入胎牛血清和L-谷氨酰胺的α-MEM培养基。每孔加入10 μL BrdU标记溶液(终浓度为10 μmol/L)后,置于37℃、5% CO2、饱和湿度的细胞培养箱中继续培养2 h。弃去培养液,每孔加入200 μL FixDenat溶液,室温孵育30 min后,弃去。每孔加入连接有过氧化物酶的BrdU单抗(anti-BrdU-POD)100 μL,室温孵育90 min后,弃去。使用PBS洗3遍,弃去PBS,每孔加入100 μL TMB(四甲基联苯胺)底物溶液室温孵育5~30 min,直至显色足够进行检测为止。置于BioTek Synergy HT多功能微孔板检测仪上,测定每孔在370 nm和492 nm波长处的吸光度值。
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根据Platinum®SYBR® Green qPCR Super Mix-UDG with ROX试剂盒说明书,用qRT-PCR仪进行qRT-PCR反应。反应条件:50℃ 2 min,94℃ 3 min;94℃ 15 s,60℃ 20 s,72℃ 1 min,40个循环。采用2-△△Ct方法[14]来评价目的基因mRNA的表达水平。靶基因引物由Invitrogen公司合成,序列见表 1。
基因 前导链(5’-3’) 后随链(5’-3’) ALP TCAGGGCAATGAGGTCACATC CACAATGCCCACGGACTTC Runx2 CGGCCCTCCCTGAACTCT TGCCTGCCTGGGATCTGTA M-CSF CATCGAGACCCTCAGACATT GCTGCTTCTTTCATCCAGTC RANKL CCAAGATCTCTAACATGACG CACCATCAGCTGAAGATAGT OPG ACAGAGACCAGGAAATGGTG CTCTCCATCAAGGCAAGAAG β-Actin GAGACCTTCAACACCCCAGCC AATGTCACGCACGATTTCCC 注:表中,ALP:碱性磷酸酶;Runx2:成骨特异性转录因子2;M-CSF:巨噬细胞集落刺激因子;RANKL:核因子кB受体活化因子配体;OPG:骨保护素;β-Actin:β肌动蛋白。 表 1 成骨细胞功能靶基因引物序列
Table 1. The primer sequences of target genes in osteoblasts
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用哺乳动物细胞总蛋白抽提试剂裂解细胞提取蛋白。使用二喹啉甲酸(BCA)蛋白浓度测定试剂盒测定蛋白浓度。使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分离抽提蛋白,在转印槽内进行半干转,将目的蛋白转到硝酸纤维素膜上,4℃封闭过夜,分别用各靶蛋白的一抗与相应二抗孵育、洗脱及化学发光显色,最后用BIO-RAD Chemi-DocTM XRS+成像系统检测蛋白表达。其中,兔源NICD多抗的工作浓度为1 : 500,抗兔源的NICD二抗的工作浓度为1 : 5000;鼠源β-Actin单抗的工作浓度为1 : 1000,抗鼠源β-Actin的二抗的工作浓度为1 : 5000。
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采用OriginPro 8绘图分析软件进行柱状图的绘制,用SPSS 12.0统计软件处理数据,对各细胞增殖、相关功能基因的表达实验数据进行分析,各组数据均来自正态分布总体,符合正态分布和方差齐性。使用Student-Newman-Keuls进行组间显著性差异分析,并对组间进行t检验,P < 0.05表示差异具有统计学意义。
NICD表达下调对辐射损伤小鼠成骨细胞系MC3T3-E1增殖和功能基因表达的影响
Effects of NICD expression downregulation on the proliferation and function-related gene expression of radiation-damaged MC3T3-E1 cells
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摘要:
目的利用RNA干扰抑制小鼠成骨细胞系MC3T3-E1表达Notch信号通路胞内结构域(NICD),探讨靶向抑制NICD表达对辐射损伤MC3T3-E1细胞的增殖和相关功能基因表达的影响。 方法建立抑制NICD表达的MC3T3-E1细胞株,利用实时定量PCR(qRT-PCR)和Western blot法检测其NICD基因的表达。MC3T3-E1细胞和NICD RNA干扰MC3T3-E1细胞经2 Gy γ射线照射后,用BrdU掺入法和qRT-PCR法检测上述细胞的增殖及相关功能基因的表达水平。使用Student-Newman-Keuls进行组间差异分析,两组间比较采用t检验。 结果用RNA干扰技术可靶向抑制MC3T3-E1细胞表达NICD。抑制NICD表达可干扰前体成骨细胞和成骨细胞的增殖。2 Gy照射后,前体成骨细胞和成骨细胞以及NICD RNA干扰的成骨细胞的增殖明显下降,各靶细胞的相关功能基因与照射前相比的变化如下:①2 Gy照射后的前体成骨细胞成骨特导性转录因子(Runx2)表达上调,差异有统计学意义(t=2.353,P < 0.05),NICD RNA干扰的前体成骨细胞Runx2表达下调,差异有统计学意义(t=2.353,P < 0.05);②2 Gy照射后的前体成骨细胞和成骨细胞以及NICD RNA干扰的前体成骨细胞碱性磷酸酶(ALP)表达上调,差异有统计学意义(t=3.182、3.345、3.555,均P < 0.05),NICD RNA干扰的成骨细胞ALP表达下调,差异有统计学意义(t=5.045,P < 0.01);③2 Gy照射后前体成骨细胞核因子κB受体活化因子配体(RANKL)表达下调,差异有统计学意义(t=2.541,P < 0.05),成骨细胞和NICD干扰的前体成骨细胞RANKL表达上调,差异有统计学意义(t=3.299,P < 0.05;t=10.212,P < 0.01),而抑制NICD表达则发生相反变化,差异无统计学意义(t=0.765,P>0.05);④2 Gy照射后的前体成骨细胞和成骨细胞骨保护素(OPG)表达下调,差异有统计学意义(t=2.994、2.782,均P < 0.05),抑制NICD表达使前体成骨细胞OPG表达上调,差异有统计学意义(t=5.841,P < 0.01),成骨细胞OPG表达下调,差异有统计学意义(t=2.544,P < 0.05);⑤2 Gy照射后各靶细胞巨噬细胞集落刺激因子(M-CSF)表达变化趋势与RANKL表达变化情况一致。 结论在不同阶段的成骨细胞中抑制NICD表达对辐射损伤表现出的作用是不同的:①可降低前体成骨细胞和成骨细胞的增殖,对辐射损伤后的前体成骨细胞的增殖有保护作用;②可通过调节Runx2从而明显抑制辐照后前体成骨细胞分化,减少骨质丢失;③辐照后各成骨细胞不会通过RANKL/OPG/RANK系统表现出对破骨细胞功能的调节作用;④成骨细胞经过调节M-CSF表现出对破骨细胞的功能抑制作用。 -
关键词:
- Notch信号通路胞内结构域 /
- RNA干扰 /
- 辐射损伤, 实验性 /
- MC3T3-E1细胞
Abstract:ObjectiveRNA interference (RNAi) is used to inhibit Notch intracellular domain (NICD) expression in MC3T3-E1 cells. The aim of RNAi is to observe the effect of the inhibition of the NICD expression on cell proliferation and function-related gene expression in MC3T3-E1 cells exposed to 2 Gy radiation. MethodsThe MC3T3-E1 cells were established to inhibit the NICD. The NICD expression of cells was detected by using qRT-PCR and Western blot. MC3T3-E1 and inhibited NICD MC3T3-E1 cells were irradiated with 2 Gy. Then, the proliferation and function-related gene expression were detected through BrdU incorporation and qRT-PCR. ResultsNICD expression in MC3T3-E1 cells could be inhibited by the RNAi technology. The inhibition of NICD expression could interfere with the proliferation of precursor osteoblasts and osteoblasts. The proliferation of precursor osteoblasts, osteoblasts, and NICD RNAi osteoblasts significantly decreased after 2 Gy irradiation. The function-related gene expression of each target cell is as follows. ① The expression of Runt-related transcription factor 2 (Runx2) was upregulated in precursor osteoblasts (t=2.353, P < 0.05) and downregulated in the NICD RNAi precursor osteoblasts (t=2.353, P < 0.05) after 2 Gy irradiation. ② The expression of alkaline phosphatase (ALP) was upregulated in precursor osteoblasts and osteoblasts and the NICD RNAi precursor osteoblasts (t=3.182, 3.345, 3.555, all P < 0.05) and was downregulated in the NICD RNAi osteoblasts (t=5.045, P < 0.01) after 2 Gy irradiation. ③ The expression of receptor activator of nuclear factor κB ligand (RANKL) was downregulated in precursor osteoblasts (t=2.541, P < 0.05) and was upregulated in osteoblasts and NICD RNAi precursors osteoblast (t=3.299, P < 0.05; t=10.212, P < 0.01) after 2 Gy irradiation. However, the inhibition of the NICD expression could cause an opposite change in other cells (t=0.765, P > 0.05). ④ The expression of osteoprotegerin (OPG) was downregulated in precursor osteoblasts and osteoblasts (t=2.994 and 2.782, P < 0.05) after 2 Gy irradiation. However, the inhibition of the NICD expression could cause expression upregulation in precursor osteoblasts and expression downregulation in osteoblasts (t=5.841, P < 0.01). ⑤ The expression of macrophage-colony stimulating factor (M-CSF) in the target cells exhibited the same trend as the expression of RANKL after 2 Gy irradiation. ConclusionsThe inhibition of the NICD expression exerts different effects on the differentiation of irradiated osteoblasts. The inhibition of the NICD expression could cause a series of changes including:①It may decrease the proliferation of precursor osteoblasts and osteoblasts and protect the proliferation of differentiating precursor osteoblasts after irradiation. ②It can significantly inhibit the differentiation of precursors osteoblasts after irradiation and reduce bone loss through regulating the expression of Runx2. ③The osteoblasts did not show the regulated function of the osteoclasts through the RANKL/OPG/RANK system. ④The osteoblasts can exhibit the inhibited function of the osteoclasts through the expression of M-CSF. -
表 1 成骨细胞功能靶基因引物序列
Table 1. The primer sequences of target genes in osteoblasts
基因 前导链(5’-3’) 后随链(5’-3’) ALP TCAGGGCAATGAGGTCACATC CACAATGCCCACGGACTTC Runx2 CGGCCCTCCCTGAACTCT TGCCTGCCTGGGATCTGTA M-CSF CATCGAGACCCTCAGACATT GCTGCTTCTTTCATCCAGTC RANKL CCAAGATCTCTAACATGACG CACCATCAGCTGAAGATAGT OPG ACAGAGACCAGGAAATGGTG CTCTCCATCAAGGCAAGAAG β-Actin GAGACCTTCAACACCCCAGCC AATGTCACGCACGATTTCCC 注:表中,ALP:碱性磷酸酶;Runx2:成骨特异性转录因子2;M-CSF:巨噬细胞集落刺激因子;RANKL:核因子кB受体活化因子配体;OPG:骨保护素;β-Actin:β肌动蛋白。 -
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